African trypanosomes present several features of interest to cell biologists. These include: a repressible single mitochondrion with a large mass of mitochondrial DNA, the kinetoplast; a special organelle, the glycosome, which houses the enzymes of the glycolytic chain; a surface coat of variable glycoprotein which enables the parasite to evade the mammalian host's immune response; and a unique flagellum-to-host attachment mechanism associated with novel cytoskeletal elements. Trypanosome development during the life cycle involves cyclical activation and repression of genes controlling these activities. Understanding the complexity of parasite development in the tsetse fly vector is especially challenging but may help to suggest new methods for the control of trypanosomiasis.

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In cultures of tsetse proboscis stages during the development of Trypanosoma congolense, attached epimastigote forms multiply and give rise to free nondividing metacyclic trypanosomes. Prevention of attachment by shaking the cultures or by providing a polypropylene substratum does not inhibit epimastigote division but does prevent the differentiation of metacyclics. We conclude that epimastigote attachment forms a necessary part of the program of metacyclic development.

Laminitis is a major cause of lameness in dairy cattle, and is widely attributed to a defect in the horny tissue that gives the hoof its mechanical strength. Defective horn is associated with, and may be preceded by, impaired keratin deposition in the hoof epidermis. The cause of abnormal keratin deposition is not easily identified but, like epidermal keratinization in other tissues, is likely to be controlled by hormones and the paracrine action of locally produced growth factors. The hormonal regulation of keratin synthesis and cell proliferation in the bovine hoof was studied using tissue explants in organ culture. As the highest incidence of laminitis is in early lactation, the study focused on insulin, cortisol and prolactin, three hormones implicated in lactogenesis and galactopoiesis. Incubation of tissue explants for 24 h in medium containing insulin (10–5000 ng/ml) stimulated protein synthesis measured by incorporation of 35 S-labelled amino acids. Histochemical examination showed that insulin binding co-localized with the site of protein synthesis. Insulin also stimulated DNA synthesis, an index of cell proliferation, which was measured by incorporation of [3H]methyl thymidine. Cortisol (10–5000 ng/ml) decreased protein synthesis, whereas prolactin (10–5000 ng/ml) had no significant effect on protein or DNA synthesis. Epidermal growth factor (10–200 ng/ml), a potent inhibitor of keratinization in other tissues, stimulated protein synthesis compared with untreated controls. Epidermal growth factor binding was located microscopically to the germinal and differentiating epidermal layers. SDS-PAGE and fluorography showed that the population of proteins synthesized in the presence of any hormone or growth factor combination did not differ from that in untreated controls and included the keratins involved in horn deposition. The results show that bovine hoof keratinization is under endocrine and growth factor control, and suggest that systemic changes in lactogenic hormones may act to inhibit keratin deposition.

Lameness is a major welfare concern in dairy cattle. Estimates of the annual incidence of lameness range from 4 to 30%, and even in well managed herds as many as 15% of animals can be affected (Esselmont, 1990). In addition to the cost in animal suffering, lameness is accompanied by loss of production on a scale comparable, in temperate countries, with that caused by mastitis. Lost production, veterinary charges and milk discard costs coupled with reduced fertility or premature culling in turn make lameness a major economic factor in dairy farming. In the UK alone, the estimated cost in lost production is £44–£90 million per annum, equivalent to £10–20 per cow (Booth, 1989; Esselmont, 1990).

The mechanical strength of the bovine hoof depends on keratinization of cells in the germinal layers of the epidermis. Histological examination of hoof tissues in calves and young heifers has identified disturbances in this keratinization process which would result in ineffective hoof development and could precipitate lameness. Short-term culture of bovine hoof tissue was used to investigate epidermal keratinization. Cell function remains viable in these cultures. The rate of protein synthesis, measured by [35S]-methionine incorporation, continued for at least 3 h in culture. Radiolabelled proteins in tissue homogenates were separated by SDS-polyacrylamide gel electrophoresis and characterised by fluorography and were representative of the proteins found in hoof tissue. Three prominent radiolabelled bands were identified as keratins and actin by Western blotting. Immunohistochemistry showed that keratin was localised principally in the epidermal layers, and microautoradiography indicated that this was the major site of protein synthesis. Hoof tissues cultured under these conditions provide a useful system for studying the acute regulation of epidermal keratinization.

Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveolus-like structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominantly in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.

Keratinization of the epidermal cells of the bovine claw generates the horn that gives the tissue its mechanical strength. Disruption of keratinization is likely to have a detrimental effect on the strength and integrity of the horn, and could lead to solar lesions and lameness. As part of a wider investigation of the cell biological causes of lameness in dairy animals, we have compared keratin synthesis and distribution in healthy bovine claw tissue with those in hooves with solar ulcers. Protein synthesis was measured by [35S]-labelled amino acid incorporation in claw tissue explant cultures. [35S]-labelled protein synthesis was higher in tissue from diseased claws than in healthy claws, and highest at the ulcer site. The identity of proteins synthesised in vitro did not differ between healthy and diseased tissue. DNA synthesis indicative of cell proliferation was also elevated in diseased tissue. Immunoblotting after one- or two-dimensional electrophoresis showed cytokeratins (CK) 4, 5/6, 10 and 14 to be amongst those expressed in healthy claw tissue. The relative abundance of these keratins was not altered in healthy regions of ulcerated hooves, nor at the ulcer site, but CK16, not usually found in healthy tissue, was detected in the sole of diseased claws. CK5/6 and CK14 were shown by immunohistochemistry to be present in the basal epidermis of healthy tissue, whereas CK10 was found in supra-basal layers. In healthy tissue from ulcerated claws, this distribution was unaltered, but at the site of solar ulcers, CK5/6 and CK14 were each found in both basal and supra-basal epidermis. The study suggests that solar ulceration of the bovine claw is not associated with gross alteration in the keratin composition of the tissue, but causes abnormal distribution of cytokeratins, perhaps as a result of loss of positional cues from the basement membrane. Ulceration did, however, stimulate cell repair involving epidermal protein synthesis (including keratins), and keratinocyte proliferation.